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REDISTRIBUTION OF BLOOD VOLUME DURING THE
ONSET OF DEOXYCORTICOSTERONE ACETATE-SALT
HYPERTENSION

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BRIDGET MAHON SEITZ

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5/08 K:IProj/Acc&Pres/ClRC/Date0ua.indd

REDISTRIBUTION OF BLOOD VOLUME DURING THE ONSET OF
DEOXYCORTICOSTERONE ACETATE-SALT HYPERTENSION

By

Bridget Mahon Seitz

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Pharmacology and Toxicology

2010

ABSTRACT

REDISTRIBUTION OF BLOOD VOLUME DURING THE ONSET OF
DEOXYCORTICOSTERONE ACETATE-SALT HYPERTENSION

By

Bridget Mahon Seitz

The splanchnic veins are known to hold the largest amount of blood within the
circulation. Structural and/or neurohormonally mediated changes in the diameter
of these vessels can lead to a reduction in splanchnic vascular capacitance and blood
volume. This causes an increase in cardiac filling pressure as blood is translocated
towards the heart. The resulting redistribution of blood into the arterial circulation
could be a factor in the development hypertension. Previous data suggests that a
reduction in vascular capacitance may play a pivotal role in the pathogenesis of
hypertension in the DOCA-salt model, but no direct measurement of volume shifts
have been reported. The purpose of my research is to assess blood volume
redistribution during the onset of DOCA-salt hypertension using bioimpedance
measurement. Regional bioimpedance allows for the measurement of total fluid
content in specific body compartments. For this study I developed a method for
repeated measurements of regional bioimpedance in conscious, unrestrained rats
over several weeks. Using this technique, I am able to show an increase in
impedance in the abdominal region in DOCA-salt treated rats (most likely indicating
a decrease in fluid content). I conclude that fluid translocation from the abdominal
region due to decreased venous capacitance may participate in the development of

DOCA-salt hypertension

Dedication

To my Mom, thank you for all your loving support and wonderful
weekly visits. You will never know how much it meant to me.

To my dear, Dad, who never was able to see the completion of this
degree. I finished for you!

To my wonderful children: Mitchell, Natalie and Cameron, thank you for
your many hugs and best of all your laughter. Being your mom will
always be my best job.

To my best friend and husband, Ted, thanks for always being my rock.
Your continual patience and confidence in me made this possible. I
could never have done it without you!

iii

Table of Contents

List of Figures .................................................................................................................. v

Introduction
1. Hypertension .................................................................................................. 1
2. Overview of the Circulatory System ............................................................. 3
3. Blood volume distribution in the circulation ............................................. 6
4. Blood volume and arterial pressure regulation .......................................... 8
5. Sympathetic nervous system activity to the splanchnic vascular bed in

hypertension .................................................................................................. 13

6. The use of bioimpedance to estimate blood volume distribution .............. 15
7. Animal model: Deoxycorticosterone acetate-salt hypertension ................ 18
8. Summary and overall importance of current research .............. ' ................. 21

Central HypothesnsZ4

Specific A1msZ4

Research Design and Methods

1. Animals ....................................................................................................... 25
2. General anesthesia ..................................................................................... 25
3. Analgesia and antibiotic prophylaxis ....................................................... 25
4. DOCA-salt hypertension protocol ............................................................. 26
S. Bioimpedance electrode implantation ..................................................... 26
6. Bioimpedance measurements .................................................................. 27
7. Radiotelemetry transmitter implantation .............................................. 28
8. Arterial pressure measurements ............................................................. 28
9. Celiac ganglionectomy .............................................................................. 29
10. Confirmation of regional denervation ..................................................... 29
11. Plasma volume measurements ................................................................ 3O
12. Animal euthanasia ..................................................................................... 30
13. Statistical methods .................................................................................... 31
Experimental Protocols and Results
Specific Aim 1 ................................................................................................... 32
Specific Aim 2 ................................................................................................... 37
Specific Aim 3 ................................................................................................... 42
Overall Discussion and Conclusions ...................................................................... 47
References ................................................................................................................... 60

iv

Figure 1:
Figure 2:
Figure 3:
Figure 4:
Figure 5:

Figure 6:

Figure 7:

Figure 8:

Figure 9:

Figure 10:

Figure 11:

List of Figures

Central Hypothesis ......................................................................... 24
Protocol for Specific Aim 2 ............................................................. 39
Bioimpedance measurement in head up postural tilt ................. 51
Bioimpedance measurement in head down postural tilt ............ 52
Bioimpedance measurement in supine position .......................... 53

Mean arterial pressure, thoracic and abdominal estimated fluid
volume in SHAM and DOCA-salt animals ...................................... 54

Tissue NE content after celiac ganglionectomy in CGX and SGX
animals ............................................................................................. 55

Mean arterial pressure, thoracic and abdominal estimated fluid
volume in DOCA CGX and DOCA SGX animals ............................... 56

Mean arterial pressure, thoracic and abdominal estimated fluid
volume in surgery and non-surgery animals ................................ 57

Preliminary plasma volume data in DOCA-salt SHAM-CGX rat...58

Initial plasma volume in surgery and non-surgery animals ........ 59

Intmductinn

1. Hypertension

Hypertension is defined as a sustained elevation in systemic arterial pressure with a
systolic blood pressure >140mmHg and/or a diastolic blood pressure of >9OmmHg.1
The World Health Organization estimates that nearly 1 billion people have
hypertension worldwide. This number is predicted to increase to 1.5 billion by
2025.2 The global disease burden attributable to hypertension is substantial
because hypertension plays a major etiologic role in the development of other
cardiovascular conditions}:3 It has been shown that even small increments in blood
pressure are associated with increased target-organ morbid outcomes, such as
stroke, coronary artery disease, renal disease and peripheral vascular disease.3 As
a result, the threshold values for defining hypertension are ever evolving. In fact,
the Seventh Report of the Joint National Committee on the Prevention, Detection,
Evaluation, and Treatment of High Blood Pressure has introduced a new category,
“pm-hypertension,” to describe individuals with blood pressure values as low as
120/80 mmHgJ-3 Most importantly, pre-hypertension is frequently considered a
“precursor" to hypertension.4 Alterations in cardiovascular structure and function
have been shown in recent studies to precede the finding of elevated blood
pressure.4 These findings have included the occurrence of left ventricular
hypertrophy,‘*'6 diastolic filling abnormality,6 endothelial dysfunction,7 as well as
impairment in autonomic regulation8 as precursors to hypertension. Whether pre-
hypertension alone, or association with its common cardiovascular risk factors,

leads to hypertension is still uncertain. However, these results provide evidence

that mechanisms operating during the early development of hypertension could be

crucial in producing the stable form of the disease.

While there is no cure for high blood pressure, it is controllable by many different
behavioral and pharmacological interventions.1 Thus, managing high blood
pressure is a lifetime commitment, adding greatly to its economic impact on society.
Further insight into the pathophysiological processes that occur during early
hypertension development may provide treatment strategies to delay or prevent the
full-blown disease and attendant target organ injury and the associated economic

burdens on society.

A single main cause for hypertension is discemable in only 5-10% of human
patients.1 Examples of such causes are renal artery stenosis, renal parenchymal
disease, and mineralocorticoid secreting tumors.9 The remaining 90-95% of
hypertensive individuals are said to have “primary” or “essential" hypertension.1
Genetic as well as behavioral factors contribute to essential hypertension,10 but to
widely differing degrees in any one individual. Thus, there are a myriad of “causes"
for essential hypertension, however all ultimately lead to a defect in the regulation
of blood pressure. Likely causes of high arterial pressure in essential hypertension
include: over activity of the sympathetic or renin-angiotensin-aldosterone systems;
renal dysfunction leading to excessive retention of sodium and water; impaired
endothelial cell release of vasodilators; and structural changes in large and small

arteries associated with aging.12

2. Overview of the Circulatory System

The regulation of arterial pressure involves many complex processes that govern
total circulating blood volume, and the function of the heart and vasculature. To
aide in understanding the potential causes of increased pressure within the arterial

system, a brief overview of the circulatory system is necessary.

The peripheral circulation (excluding the pulmonary circulation) is a semi-closed,
blood-filled circuit that consists of a high-pressure arterial side and a low-pressure
venous side arranged in series. Capillaries connect the two sides where oxygen,
nutrient and metabolite exchange occurs between blood and tissue. The venous and
arterial circulations work in balance and are controlled by neuronal and humoral
factors that help achieve overall homeostasis of the circulatory system.”13 The
main function of the arterial vessels are to distribute oxygenated blood from the
heart to peripheral organs such as the muscle, viscera and brain. In addition, small
arteries and arterioles in various organs within the circuit control regional
distribution of blood flow. This distribution by adjusting the arterial lumen
diameter through changes in the tone of the vascular smooth muscle located within
the walls of this vessels.12 In contrast, the main function of the venous system is to
collect deoxygenated blood and return it to the heart. Some veins also serve as
important blood holding (capacitance) vessels. Veins are 30 times more compliant
than arteries and therefore are capable of holding relatively large blood volumes

without alterations in their intraluminal pressure.13 In conclusion; arterial function

regulates resistance and flow through the circulation, while venous function

regulates capacitance and volume.14

Arterial blood pressure is typically described (using an electrical analogy, i.e. Ohm’s
Law) as the product of two hemodynamic variables, one directly measured and the
other calculated. Cardiac output (CO) can be directly measured as the volume of
blood that is ejected from the heart and flows through the arterial system per unit
time; it is the product of heart rate (HR) and stroke volume.” Systemic vascular
resistance (SVR) is a calculated variable that reflects the many forces within the
circulatory system that impede blood flow; the most important of these forces is the
vascular diameter of small arteries and arterioles.15 Hypertension occurs when the
value of one (or both) of these hemodynamic variables (CO and SVR) is increased

above the ”normal" range for arterial pressure.

In general, the onset of essential hypertension in humans has been shown to be
associated with an increase in CO, whereas in the established phase of hypertension
SV R is increased in most people.”17 Hence, the majority of research with regards
to the causes of hypertension have focused on mechanisms that affect either the
diameter of small arteries and arterioles, or systemic blood flow driven by the heart
(primarily total circulating blood volume or cardiac function per se. However,
arterial pressure also can be simply expressed as the blood volume within the
arterial system divided by the compliance of the arterial system18 Compliance

reflects the ability of a vessel to distend and increase volume with increasing

transmural pressure (i.e. the difference between intraluminal and extramural
pressure).13 This understanding of arterial pressure regulation does not focus on
the steady-state flow of blood through the arterial system (CO), but instead on
steady-state changes in arterial volume which can be brought about by transient
differences in arterial system inflow (determined by CO) and outflow (determined
by vascular resistance). These volume differences are well-understood to account
for the pulsatile nature of arterial pressure created by cyclic cardiac contraction and
relaxation, but are less often considered explicitly as a major determinant of the
chronic level of arterial pressure (which is key to the diagnosis of hypertension).
This is probably due to the fact that flow through the circulatory system can be
readily measured, whereas the volume of the arterial system is difficult to
determine accurately. Nevertheless, within this conceptual framework, possible
mechanisms of hypertension are either: 1) a decrease in arterial system compliance,
or 2) an increase in steady-state arterial system volume. Decreased compliance of the
aorta and large arteries is widely believed to account for the specifically elevated
systolic pressure (isolated systolic hypertension) that is often observed with aging,
and in patients with established essential hypertension. 19 Small artery compliance
is. often normal, however can even be increased in essential hypertension, or
decreased with age or in cases of severe hypertension.” However, the focus of the
research described in this thesis is on how changes in vascular volume may affect

arterial pressure.

3. Blood volume distribution in the circulation

Blood is held within all parts of the cardiovascular system, with approximately 70%
of blood volume is contained in the venous system.” “Active capacitance" veins are
filled or emptied by changes in tone of the smooth muscle within their wall.20
“Conduit" veins are mainly filled and emptied by passive forces associated with
gravity or external compression by surrounding tissue.” Therefore, either changes
in venous vascular tone (venoconstriction) or passive changes in venous diameter,

can cause dramatic shifts in blood volume within the circulation.

There are two compartments within the venous system: central and peripheral.
Importantly, veins within these two compartments are not uniform in their
compliance or capacitance.”21 Veins in the peripheral venous compartment are
very compliant and have a high capacitance.“ The splanchnic veins, located in the
abdominal region, represent the largest blood volume reservoir within the human
body”, and exhibit the greatest degree of active capacitance response of any veins
in the body.20 Veins in the central venous compartment are far less compliant”
The thoracic vena cava and the great veins of the central venous compartment are
unable to store blood volume to any great extent.” However, it is the central veins
that are critical for circulatory dynamics.21 Any stimulus that causes a decrease in
peripheral venous compartment capacitance (active or passive) will redistribute
blood from the peripheral to the central venous compartment. Because of the low
compliance of the central venous compartment, this augments the amount of blood

entering the heart (venous return). This increased volume of blood entering the

heart transiently exceeds the amount of blood leaving the heart (cardiac output),
resulting in enhanced cardiac filling pressure and volume.14 This leads, via the
Frank-Starling mechanism, (increase in pre-load results in an increase in
contractility), to augmented blood ejection by the left ventricle, and a portion of the
excess blood that entered the heart being redistributed into the systemic circulation,

including the arteries.

Changes in venous vascular capacitance can also function as an important
physiological compensatory mechanism, adjusting the circulation in response to
everyday stresses. The hemodynamic response to exercise illustrates the dynamic
changes within the venous vasculature required to respond to the requirements of
the body for blood and oxygen delivery and waste removal. During exercise, as the
artery supplies blood and oxygen to the working muscle, veins undergo both active
and passive emptying, i.e. there is a decrease in venous vascular capacitance and a
shift of “stored" blood towards the central circulation, increasing venous return (VR,
flow of blood to the heart), CO and arterial pressure.22 Similar adaptations can also
be seen under normal physiological conditions even without physical exertion. For
example, performing a challenging arithmetic problem can alter venous capacitance.
In one study, subjects performing difficult subtraction problems had an increase in
forearm venoconstriction, which caused blood to be displaced towards the heart,
increasing CO and arterial pressure.23 Thus, venous vascular capacitance is
important in the regulation of arterial blood pressure in a normal physiological state

as well as, in a pathological state, such as hypertension.

4. Blood Volume and Arterial Pressure Regulation

Despite many years of research, there is no unifying theory to account for the
pathogenesis of essential hypertension. What has been shown is that during the
early stage of arterial pressure elevation there is often an increase in central blood
volume and transient increases in stroke volume and CO.”17 Eventually, as the
disease becomes more long- standing, these hemodynamic processes revert to
physiologically normal and hypertension is continually sustained by an elevation in

systemic vascular resistance.”'15

According to this scenario, during the onset of hypertension blood volume increases
in the central venous compartment, cardiac filling rises, and the Frank-Starling
mechanism leads to more blood volume being expelled into the low compliant
arterial beds. This results in an elevation in CO, arterial volume and arterial
pressure.21 It is important to note that under normal physiological condition, the
heart and lung have relatively low and fixed blood storage capacities. Thus, in this
discussion, the pulmonary circulation is not a factor and the heart is represented as
merely a “demand" pump, which moves blood from the low pressure, high

compliant venous system into the high pressure, low compliant arterial system.

In accordance with this model, two general mechanisms could produce an increase
in central blood volume and contribute to a rise in arterial pressure. The first
mechanism is an increase in the total volume of blood contained within the entire

circulation. When total circulating blood volume increases, blood volume and/or

blood pressure increases in all vascular compartments in relation to their

compliance. 20

Total blood volume is a relatively fixed fraction of the extracellular fluid volume and
is determined primarily by the balance between sodium (the main extracellular
osmotic particle) and water intake, and renal sodium and water excretion.”25 The
major site of regulation of sodium and water homeostasis is the kidneys. Therefore,
renal function has a major influence on total circulating volume, and as a result,
arterial pressure.”'24'25 In fact, arterial blood pressure has often been used as a key

indicator of the volume of extracellular fluid within the circulation.26

Although that principle is well accepted, in the 1960’s Arthur Guyton and his
colleagues proposed an additional relationship between arterial pressure and renal
function that has been more controversial. Their proposition was that renal sodium
and water excretion are directly proportional to the prevailing level of arterial blood
pressure (the so-called “pressure-natriuresis relationship”).27'29 The implication of
this dual cause-and-effect relationship between arterial pressure and renal function
are elaborated in great detail in numerous papers and their now famous
mathematical model of the circulation.28 TheGuyton mathematical model affirms
that the level of steady-state arterial pressure is determined entirely by the ability of
the kidney to excrete sodium and water. 2330 According to this hypothesis,
hypertension can only occur when the pressure natriuresis relationship is altered in

a way that leads to the kidneys retaining more sodium and water at a given

pressure, thereby producing at least a transient increase in total blood volume in the

circulation.”31

An increase in total blood volume alone has been shown to elevate arterial pressure
and contribute to the development of hypertension.”34 This is evident in some
obese borderline hypertensive patients, which have increased total blood volume
compared to age-matched lean borderline hypertensive patients. In addition, 90%
of the cases of hypertension in patients undergoing hemodialysis for end stage renal
disease are specifically due to sodium and volume overload.36 In support of these
findings, adequate reduction and control of total blood volume achieved

normotension in these previously hypertensive hemodialysis patients.37

These examples represent specific instances where total blood volume parallels
arterial pressure. However, this is not always the case. The literature most often
reports total blood volume to be reduced in essential hypertension and inversely
associated with arterial pressure.”'16'20'21-38'41 This inverse relationship is further
supported by situations where higher than normal total blood volume occurs while
blood pressure is below normal, such as in hepatic cirrhosis” and pure autonomic
failure."3 In conclusion, total blood volume can, but is not always consistent with

arterial pressure.

The focus of my research is on a second possible mechanism to produce increased

arterial blood volume and thereby arterial hypertension: a redistribution of the total

10

blood volume within the circulation. With this mechanism, no increase in total
blood volume or impairment in renal function, is required; in fact it can operate in
the presence of a reduction of total blood volume (as is often observed in individuals
with hypertension).”'33‘40 Instead of arterial blood volume being elevated due to an
overall increase in body fluid volumes, this mechanism consists of a translocation of
blood from the highly compliant, blood storing venous circulation into the low
compliant, high pressure, arterial system. In support of this idea, earlier research
demonstrated that during the early phase of hypertension development there was a
higher fraction of the total circulating intravascular volume in the central
circulation.” This was found without any alterations in total blood volume.
Theoretically this redistribution of blood volume could be achieved by any
mechanism (for example, vigorous muscular exercise, as discussed above) that
decreases overall venous capacitance of the peripheral venous compartment, and
leads to the mobilization of blood volume into the central venous compartment and

then into the arterial system.

The two mechanisms described above that produce elevated arterial system volume
and pressure are not mutually exclusive, nor are they exhaustive. Another means to
increase arterial volume is via increased resistance in small arteries and arterioles,
which effectively “traps" an increased volume of blood in the upstream arterial
tree.” This mechanism is most often observed in established forms of
hypertension.”17 All three of these mechanisms operate together under normal

physiological circumstances, and all potentially contribute to the pathophysiology of

11

hypertension. However, the relative impact of volume redistribution on arterial

pressure regulation is often underestimated as a significant contributor.

An example of the significant influence of blood volume redistribution on arterial
pressure can be observed in supine hypertension in individuals with peripheral
autonomic failure“:3 While most of these patients have abnormally low arterial
blood pressure—due to a decrease in blood volume or an increase in peripheral
venous pooling (from gravitational force) when standing—dramatic hypertension
occurs in these, patients when supine.“ The likely explanation for the occurrence of
hypertension while patients are in the supine position is a redistribution of blood
volume from the highly compliant splanchnic veins towards the central circulation
and ultimately into the arterial system (as described earlier). This explanation is
supported by the observation that the hypertension can be successfully treated with

venodilating drugs, but not arterial dilating drugs such as nitroglycerin/*3-45

The situation in patients with autonomic nervous system failure complements
substantial other evidence indicating that, in addition to gravitational forces,
activation of the sympathetic nervous system (SNS) is a major mechanism for
redistribution of blood volume, especially by affecting splanchnic vascular

capacitance.18;20-21.29

12

5. Sympathetic nervous system activity to the splanchnic vascular bed in

hypertension

Sympathetic nervous system activity is elevated in some humans with
hypertension,29-45'48 and in several experimental hypertensive animal models”51
(for example, DOCA-salt and spontaneously hypertensive rats). The elegant work of
Esler and colleagues“'8 using norepinephrine spillover techniques revealed that
sympathetic activity was increased in specific vascular beds in human essential
hypertension: the majority of the increase was found in the heart and kidneys.
Moreover, this increase was most prominent in borderline or pre-hypertensive
subjects, which further suggests that over activity of the SNS is a key component in

the development of hypertension"!7

Splanchnic veins and venules located in the abdominal region, which directly supply
blood to the central venous compartment, are richly innervated by the SNS.21-57-'53
Activation of the SNS increases the vascular tone of smooth muscle surrounding
these highly compliant, large blood-storing veins.54 As discussed above, this allows
for mobilization of blood toward the central venous compartment and heart.
Consequently, sympathetic venoconstriction of the splanchnic veins can be an

important regulator of cardiac filling and arterial pressure.”-20'21»53-54

There is evidence of activation of the splanchnic SNS in hypertension. ”JO-215354 A
significant increase in splanchnic nerve activity, measured by direct nerve

recordings in conscious rats, was found in angiotensin II-induced hypertension.55 A

13

sympathetically mediated increase in venous motor tone was also found during the
development of angiotensin II-salt hypertension in rats.51v56 Indirect evidence
found in human borderline hypertension showed vascular resistance to be elevated
in the hepatosplanchnic circulation, (a region where sympathetic activity is often
elevated), before occurrence in other vascular beds.57 These findings suggest the
importance of sympathetically mediated constriction of splanchnic capacitance

vessels during the developmental stages of hypertension.

Splanchnic arteries and veins are innervated by sympathetic nerves through the
paravertebral and prevertebral ganglia.58 Postganglionic neurons innervating the
splanchnic vascular bed are located within the celiac and superior mesenteric
ganglia, which are fused and referenced as the celiac plexus.59 Therefore,
sympathetic input to the splanchnic vascular via the celiac plexus has the potential
to decrease splanchnic venous capacitance. In a paper from 1941, successful
treatment of human hypertension was achieved by surgically removing the celiac
ganglia (celiac ganglionectomy, or CGX).">0 In the DOCA-salt and ANG II
experimental animal models, CGX has shown to reduce arterial pressure.56 Studies
in humans with essential hypertension demonstrated that surgical section of the
splanchnic nerves also is effective in treating essential hypertension.61
Additionally, celiac plexus neurolysis for the treatment of pancreatic malignancies in
humans often results in transient but severe hypotension.62 Finally, chronic
electrical stimulation of the splanchnic nerves in dogs produced sustained

hypertension.63'64 Together these findings indicate that splanchnic

14

sympathoactivation may be an important factor in the development of hypertension.
In my experiments, CGX will be used to examine the contribution of splanchnic
sympathetic nerves to blood volume redistribution and hypertension development

in rats.

6. The use of bioimpedance to estimate blood volume distribution

Changes in total circulating blood volume can be a key factor in diseases such as
congestive heart failure“, in medical conditions such as syncope“, and possibly in
hypertension (as discussed earlier). Accurate measurement of total circulating
blood volume nevertheless is difficult and remains a clinical challenge.“68 But,
most importantly, methods used to measure total circulating blood volume do not
take into the account the distribution of blood within the circulation. The central
hypothesis of my research is that both the total amount of blood within the
circulation, and its relative distribution in high and low compliance vascular
compartments (veins and arteries, respectively), can contribute to hypertension

development.

A significant obstacle to testing this hypothesis is that there is currently no method
available that allows direct quantification of arterial blood volume or venous blood
volume. Arterial and venous volume fractions have been estimated in individual
organs using complicated plethysmographic or imaging methods69'70, however these

approaches are not applicable for use in the whole animal. Instead, investigators

15

 

have taken advantage of the fact that venous blood volume is much larger than
arterial volume in all tissues, and that some vascular regions (splanchnic organs)
have much larger blood storing capacities than others (muscle, skin, heart .1430-
21495653 Thus, with a few exceptions (e.g. heart) a measured decrease in organ size,
weight, or total blood content is generally accepted to reflect primarily a decrease in
venous blood volume. And as discussed earlier, the high capacitance splanchnic
organs are understood to represent the bulk of the “peripheral” venous
compartment. Therefore, measurements of regional blood volume (especially in the
splanchnic organs) are used as a surrogate for direct measurement of venous

volume.

Blood volume distribution within multiple compartments of the circulation can be
determined in animals and humans using a variety of techniquesé7-63-71, but the best
approach currently available is blood pool scintigraphy.72 This method allows for
direct tracking of radiolabeled blood cells in various vascular regions. However, this
approach does not offer repeated or continuous measurement of changes in blood
volume over extended periods of time in the same animal. That is why for these
studies I chose to use bioimpedance, a well-established method to estimate changes
in vascular volume within specific compartments of the circulation.73'74 Specifically,
this method offers the possibility to make continuous (or repeated) measurements

in conscious rats.

16

 

Bioimpedance methodology is based on a simple model that regards the body
(human or animal) as a cylindrical conductor, composed of various electrical
compartments comprising simple resistors and capacitors.75 Bioimpedance is
measured by the conductive response of the body to an externally applied low
voltage electrical current and the resulting resistance or impedance to this
current.”""5 Bioimpedance is inversely proportional to the estimated amount of
total body water present74v76'77 As impedance increases, the volume of estimated

total body water decreases and vice versa.

Segmental bioimpedance can be used to monitor fluid volume changes within
specific anatomic regions.76 Moreover, the measurement of bioimpedance is
routinely used as a surrogate for shifts in blood volume.”80 Total body water is
composed of both extracellular and intracellular fluid. Intracellular fluid is rarely
mobile within the circulation. Thus it is the extracellular fluid, composed mainly of
blood and interstitial fluid that can be readily mobilized within a body region. Blood
volume in most regions of the body is much larger than the interstitial fluid volume.
Therefore, changes in impedance can be used as a proxy for changes in blood
volume.74'75 Thus, our hypothesis was addressed using the technique of
bioimpedance. For this research, the anatomical regions used for segmental
bioimpedance were: (1) the chest, representing the central venous compartment,
heart and lungs; and (2) the abdomen, representing the splanchnic region, which is

a component of the peripheral venous compartment

17

Although bioimpedance has been extensively validated in humans, the application of
bioelectrical impedance in experimental animal models has been sparse, especially
in conscious unrestrained animals. In order to utilize this technique, the technique
would need to be used in conscious and free-moving laboratory rats to make
repeated, within-animal estimates of blood volume redistribution between the

peripheral (splanchnic) and central venous compartments.

7. Animal Model: Deoxycorticosterone acetate-salt Hypertension

Over the past 50 years, numerous experimental models of hypertension have been
developed, predominantly in rats.50 Because the etiology of essential hypertension
is heterogeneous, it is nearly impossible for any one experimental rat model to
encompass all facets of this disease. For this study, I chose to use the
deoxycorticosterone acetate (DOCA)-salt hypertensive rat model, which has been
shown to be an effective model to study volume-dependent hypertension. This
hypertensive model involves the chronic treatment of rats with the
mineralocorticoid, DOCA, in combination with a high salt (1% NaCl) intake via the
drinking solution, and unilateral nephrectomy. The model is considered a
prototypical example of salt-sensitive hypertension.81 The concept of salt-
sensitivity refers to the finding that some individuals exhibit an exaggerated
increase in blood pressure when consuming high dietary salt, whereas others show
little change in blood pressure.82 The former individuals are referred to as ‘salt-

sensitive’. Thus, the concept of salt sensitivity provides an explanation for the

18

generally reproducible connection between high salt intake and elevated blood
pressure in large populations of people in which many individuals have normal

blood pressure despite daily consumption of large amount of salt.

DOCA is a potent mineralocorticoid that is known to cause significant sodium and
water retention83 by reducing the ability of the kidneys to excrete sodium.“ The
effectiveness of DOCA in elevating blood pressure is directly proportional to the salt
intake of the animal.“ Sodium is the major cation in the extracellular fluid and its
excretion is regulated almost exclusively by the kidney.27 The level of total body
sodium, which is increased in the DOCA-salt model, contributes significantly to the
osmolality and volume of the extracellular fluid. Thus, the regulation of total body
sodium has become synonymous for volume regulation.” Therefore, DOCA—salt has

commonly been classified as a volume-dependent model of hypertension.85

It is widely accepted that DOCA-salt treatment causes an increase in total body
water with prOportional increases in circulating fluid volume.“ Furthermore,
recent work in dogs with renal hypertension86 shows that total body water is a
major determinant of arterial pressure. Thus, it is axiomatic that DOCA-salt
treatment produces an elevation in arterial blood pressure by increasing total fluid

volume within the circulation.

Nevertheless, recent data has revealed that total body water is not always directly

associated with arterial pressure. For example, Titze et a131-97'88 have shown that

19

sodium retention can take place free of water accumulation, including in DOCA-salt
hypertension in rats. High sodium intake in DOCA-salt treated rats resulted in an
increase in total body sodium content by 50% within 5 weeks; however only ~ 20%
of the sodium accumulated led to volume retention.87 As a result, only moderate
increases in total body water was achieved despite massive sodium retention. The
excess sodium was shown to be stored in osmotically inactive and/or osmotically
neutral sodium storage sites, which did not play a part in volume expansion, yet the
animals were hypertensive.”88 These results argue against the traditional volume-
dependent view held of the DOCA-salt model of hypertension, and provide clear
evidence that an overall increase in total body sodium does not always increase

arterial pressure through blood volume expansion.

It has also been shown that DOCA-salt increases venomotor tone by activation of
the SNS.49'54'85 As described earlier, the SNS is an active regulator of splanchnic
venous capacitance and thereby contributes to the redistribution of blood volume
from the peripheral to the central venous compartment. A study comparing DOCA-
salt rats to sham-operated rats revealed no difference in total circulating fluid
volume between the two groups yet the DOCA-salt rats were hypertensive.85 It can
be speculated from this study that DOCA-salt treated rats had an increase in central
blood volume due to a decrease in peripheral venous capacitance, although this was
not measured directly. In a study in sheep, however, mineralocorticoid-salt

treatment caused an immediate and sustained increase in central venous pressure

20

and stroke volume without any change in total body fluids, strongly suggestive of

peripheral to central blood volume redistribution. 89

In a well-devised study, the experimental question should dictate the experimental
model.50 The DOCA-salt experimental rat model offers this opportunity to address
the experimental question of vascular volume, and its regional redistribution affect

on arterial pressure regulation.

8. Summary and Overall Importance of Current Research

The overall hypothesis of this work is that volume redistribution within the
circulatory system (from peripheral towards central venous compartment and into
arteries) affects arterial pressure during the onset of hypertension. In order to
address this aim, the DOCA-salt experimental rat model and the technique of
segmental bioimpedance was used. The DOCA-salt model has long been cited as a
volume-dependent model of hypertension. (i.e. increased blood pressure is caused
by increased body fluid volume.) Overall volume expansion within the circulatory
system, presumably caused by alterations in the pressure-natriuresis mechanism, is
well documented as contributing to the regulation of arterial pressure.2‘”7-29
However, studies have also shown that the initial elevation of arterial pressure in
early stage essential hypertension may occur by another mechanism—a

redistribution of fluid volume—which is independent of overall circulatory volume

expansion.”'20‘21-49 This translocation of blood volume from the peripheral to

21

central venous compartment is likely brought about by a decrease in peripheral
venous capacitance due to activation of the SNS, specifically within the

splanchnic veins. ”-14.182021.”

Segmental bioimpedance was used to estimate changes in blood volume between
the peripheral and central venous compartments in conscious unrestrained rats
treated with DOCA and salt. Unlike other methods to determine blood volume
distribution, the bioimpedance method allows for repeated (or continuous)
measurement of regional blood volume within the same animal over an extended
time. Tracking fluid movement during the transition from normal blood pressure to
hypertension and back to normal pressure could provide significant insight into the
role of blood redistribution (and the mechanism driving this movement, presumably
increased sympathetic tone to the splanchnic veins) in the development of
hypertension. Furthermore, I purpose that this was due to an increase in

sympathetic tone to the splanchnic veins.

My research is important as it provides additional insight into one possible
mechanism, blood volume redistribution, which has been cited in contributing to the
development of hypertension, but no direct evidence has been shown to support it.
It is well established that hypertension is the leading cause of cardiovascular death
and disability worldwide. This is forecasted to continuel'2 (by 2025, 1.5 billion
people are predicted to be hypertensive.)2 Thus, there is much to be gained by a

further understanding of the mechanisms that occur during the early development

22

of hypertension. This is crucial in delaying or preventing the stable form of this
continuous disease, as well as, reducing end organ damage and the significant

economic burden that accompanies this lifetime condition.

23

SW
A factor in the development of DOCA-salt hypertension is reduced splanchnic
vascular capacitance leading to blood volume redistribution from the high-

compliance splanchnic vasculature (abdomen) toward the central circulation (heart

and lungs), and ultimately into the less compliant arteries.

/ MAP

 

h
\

A
\ SVR -/

\.,—../

Figure 1: The circulatory system is a semi-closed loop. The volume and pressure
contained within the arterial system, as well as, the systemic vascular resistance
(SVR) are all components in determining the mean arterial pressure (MAP).

5 'fi 5'
1. Specific Aim 1: To establish a bioimpedance method in a conscious reely

moving laboratory rodent to estimate circulatory fluid volume shifts.

II. Specific Aim 2: Use chronic bioimpedance measurements to determine the

changes in blood volume during the onset of DOCA-salt hypertension.

III. Specific Aim 3: Determine if activation of the sympathetic nervous system
contributes to redistribution of blood volume during the development of

DOCA-salt hypertension.

24

Weds

1. Animals
Male Sprague-Dawley rats (Charles River Laboratories, Portage MI) weighing 300-

325 grams and normotensive at the beginning of the study were used in all
experiments. The Michigan State University Committee on Animal Use and Care
approved all protocols. The rats were housed in groups of 3 in Plexiglas® cages in a
temperature and humidity controlled room with a 12-hour light/dark cycle and
allowed to acclimatize for a week prior to any surgical procedures. Rats were given
standard pelleted rat chow (8640 rodent diet; Harlan/Teklad, WI) and water ad

libitum.

2. General Anesthesia

All surgical procedures were performed using an inhalation anesthetic agent
(isoflurane) delivered through a nose cone. The induction chamber contained a 4%
isoflurane in oxygen mixture. The animals were maintained during the surgery by
2% isoflurane in oxygen mixture. Rats were recovered from anesthesia under close

observation on a heating pad.

3. Analgesia and Antimicrobial Prophylaxis

Post-operative analgesia and antimicrobial prophylaxis were established by
administration of enroflaxcin (5 mg/kg IP), ticarcillin-clavulanate (200 mg/kg IP)
and carprofen (5 mg/kg SQ) respectively. Post-surgery analgesia was continued for

an additional 5 days with carprofen (5mg/kg SQ).

25

4. DOCA-salt Hypertension Protocol

All rats underwent a unilateral nephrectomy of the left kidney to reduce renal mass
before the start of the experiment Under general anesthesia, a left lateral
abdominal incision was used to access the left kidney. The left kidney was
exteriorized, left renal vessels and ureter were tied off, and left kidney was
removed. The incision was sutured closed in layers. Following 7 days of surgical
recovery and a 5-day control period, all rats were randomly divided into their
respective groups. Rats that were to receive a DOCA pellet were allowed free access
to saline (distilled water containing 1% NaCl and 0.2% KCl) for 3 days. Sham-
operated rats (SHAM) were allowed free access to distilled water throughout the
study. After 3 days, a DOCA pellet (150 mg/kg) impregnated with silicone rubber
was implanted subcutaneously on the dorsal side of the DOCA-treated group of rats
while the SHAM group underwent a similar subcutaneous surgery but did not
receive an implant. After 5 days, the DOCA-treated group were taken off saline
drinking solution and given free access to distilled water for the remainder of the

experiment.

5. Bioimpedance Electrode Implantation

The bioimpedance technique was achieved by permanently implanting 6 stainless
steel electrodes (Plastics One, VA) subcutaneously. While rats were under general
anesthesia, electrodes were implanted subcutaneously through a 1 cm incision
made in the skin above the specific anatomical location as described below. The
electrodes were tied into the underlying muscle with suture and the incision was

sutured closed. All the electrodes were exteriorized between the scapulae and

26

were connected to a swivel arm that allowed the rat free-movement throughout the
cage. All electrodes were in place before the start of the experiment. The rats were

given 7 days to recover from electrode implantation.

6. Bioimpedance Measurements

Bioimpedance was used to detect internal volume shifts chronically in conscious,
free moving rats. The electrodes were connected to a two-channel‘tetrapolar high
frequency impedance meter (Thrim®) that introduced a high frequency, low
amperage, constant current, which was insensible to the rat. Current was injected
into 2 electrodes positioned dorsally at the base of the head and tail. These
electrodes represent the driving electrodes through which current (excitation:1mA
and frequency:51.2kHZ)) is injected into the rat. In addition, 4 electrodes implanted
at precise pair-wise distances across the thorax (midline xiphoid process), and
abdomen (iliac crest) represents the detecting electrodes, which measure the

voltage drop across these anatomical segments of interest: thorax and abdomen.

Impedance or resistance to this high frequency current is inversely proportional to
the amount of fluid volume in the tissues between the recording electrodes. As
compartmental fluid volume decreases, measured impedance increases. Each rat
was measured for abdominal and chest impedance daily for a duration of 20
minutes at the same time every morning while housed undisturbed in their home
cage. An average for the recorded 20 minutes, for both abdominal and chest
impedance, were derived by a commercially available data acquisition program

(Powerlab, ADinstruments) per rat. Each daily impedance average was converted to

27

estimated compartmental fluid volume value by the following mathematical
conversion: 10 + 1/bioimpedance recording, which was used to avoid very small
values. The inverse of bioimpedance is sometimes referred to in the literature as
“admittance”. Bioimpedance and impedance are used interchangeable throughout

this thesis.

7. Radiotelemetry Transmitter Implantation:

Radiotelemeter transmitters for measuring arterial pressure were implanted in all
rats before the start of the experiment. Under general anesthesia, the tip of the
transmitter catheter was inserted into the abdominal aorta by way of the femoral
artery. The body of the transmitter was placed in a subcutaneous pocket along the
abdomen but below the abdominal impedance electrodes, so as not to interfere with
bioimpedance measurements. The incision was closed in layers. All animals were

given 7 days to recover.

8. Arterial Pressure Measurements

Rats were housed in individual Plexiglas® cages and placed on radiotelemetry
receivers (RPC-l, DSI). The arterial blood pressure was recorded for 10 seconds
every 10 minutes throughout the experiment. A commercially available
radiotelemetry data acquisition program (Dataquest, DSI) was used to remotely
monitor the arterial pressure. The radiotelemetry system did not interfere with

bioimpedance recordings during the study.

28_

9. Celiac Ganglionectomy

Under general anesthesia, a ventral midline abdominal incision was performed and
the small intestines were gently retracted and placed on warm ticarcillin-
clavulanate (200mg/kg) and enroflaxcin (5mg/kg) soaked gauze. The celiac plexus
located between the aorta, celiac artery and mesenteric artery was dissected free
and removed. The small intestines were placed back into the abdominal cavity and
lavaged with warm saline. The midline abdominal incision was sutured closed in
layers. The SHAM group underwent a sham operation that was performed by

accessing and exposing the celiac plexus only.

10. Confirmation of Regional Denervation

At the completion of the protocol the rats were sacrificed by an intraperitoneal
injection of sodium pentobarbital (100mg/kg). The liver, spleen, small intestines
and right kidney, which represent organs that are innervated by the celiac ganglia in
the splanchnic region, were harvested from each rat The tissues were weighed and
immediately frozen in liquid nitrogen. All tissues were stored at -800C for later
analysis. High performance liquid chromatography (HPLC) analysis was used to
measure norepinephrine (N E) content in each tissue, as an index of the density of
sympathetic innervation of the tissues. Data was reported as nanogram of NE per
gram of tissue. The Michigan State University Department of Pharmacology and

Toxicology HPLC core facility performed these assays.

29

11. Plasma Volume Measurements

All rats were chronically instrumented with silicone-tipped catheters into the
femoral artery and vein for Evans blue administration and blood sampling. The two
catheters were exteriorized between the scapulae of the rat into a stainless steel
spring that was attached to the rat at one end by a loosely fitted rubber jacket
(Instech Solomon), and the other end to a swivel arm at the top of the cage. This
allowed for free movement and access to the catheters without handling or
disturbing the rat. The rats were housed in individual Plexiglas® cages and
recovered for 5 days. All catheters were flushed daily with a heparin-saline
solution. Plasma volume was estimated with the use of 10-minute distribution
volume of Evans blue dye. Arterial blood (0.6ml) was collected at baseline (before
Evans blue dye injection) and 10 minute after intravenous Evans blue dye (1mg/ml
in saline) injection. The Evans blue dye will remain in the vascular space during this
timeframe. All blood samples were collected in heparinized EDTA tubes and
centrifuged. The plasma was collected. Evans blue dye concentration was
determined by spectrophotometry and absorbance was read at 650nm. The dye
concentrations in the collected samples were measured by using a standard curve of

Evans blue dye solution in the plasma of the baseline sample.

12 Animal Euthanasia

At the conclusion of each study, an intraperitoneal injection of sodium pentobarbital
(100 mg/kg, i.p.) was administered. This adheres to the Michigan State University

Animal Care and Use Committee guidelines for euthanasia.

30

13 Statistical Methods

Mean arterial pressure and impedance data were statistically analyzed by one-way
repeated measures ANOVA. When applicable, post-hoc multiple comparisons using
Dunnett’s procedure (GraphPad, Instat) was used to compare all days to control
period day 2. A p-value <0.05 was considered significant. All results are reported as

mean _+_ SE.

31

S 'fiE . Hill I II! II

I. Specific Aim 1: To establish a bioimpedance method in a conscious
unrestrained laboratory rat to estimate circulatory fluid volume shifts

during the onset of hypertension.

W:

Bioimpedance is a novel technique used to determine estimated fluid volume
changes within specific regional vascular compartments.73'74'76 In this study, I used
bioimpedance to measure changes in fluid volume within the abdominal region,
representing the highly compliant splanchnic vascular compartment, and the
thoracic region, representing the lesser compliant central vascular compartment.
These measured changes in fluid volume were used as a proxy for blood volume
shifts into and out of these vascular regions. 74-30 Although other methods of blood
volume distribution are available, bioimpedance was used because it allows for

repeated measures in individual animals.

It is well documented that gravity, as illustrated by cardiovascular responses
produced by postural changes (i.e. tilting), is a potent regulator of blood volume
distribution within the vascular system.”'15'9°'92 During passive postural tilts, the
head is moved above (head up tilt; HUT) or below (head down tilt; HDT) the level of
the heart During initial changes in posture, blood volume is shifted between the
peripheral and central (heart and lung) vascular compartments by gravitational
forces; later, compensatory mechanisms (e.g. baroreceptor reflex and neurohumoral

factors) also contribute to maintaining homeostasis of the cardiovascular

32

system.”93 Therefore, postural tilting is one reliable way to induce blood volume
shifts between specific vascular compartments. I used this approach in order to

validate the bioimpedance technique in rats.

During passive head-up tilt (HUT) gravity pulls blood out of the chest towards the
compliant veins of the abdomen and lower extremities. As a result, central blood
volume is reduced. This leads to a decrease in venous return and cardiac output,
resulting in a transient lowering of mean arterial pressure.”-92'93 The unloading of
arterial baroreceptors causes sympathetic vasoconstriction of veins and arteries in
the peripheral compartment The result is reduced peripheral compartment
capacitance and redistribution of blood back towards the central venous

compartment, heart and arteries to maintain arterial volume and pressure.

During HUT in rats I anticipated an initial increase in impedance in the chest
(decrease in fluid volume) as blood moves out due to gravitational change.
Impedance in the abdomen should decrease (increase in fluid volume), as blood
gathers in the veins of the large capacity splanchnic region and lower limbs. These
impedance changes should eventually stabilize due to activation of compensatory

mechanisms to maintain cardiovascular homeostasis.

During head-down tilt (HDT), gravity works to move blood from the abdomen and
other peripheral vasculature into the central venous compartment. This increases
central venous compartment volume, venous return and cardiac output resulting in

a transient increase in arterial pressure.”-‘”93 The cardiovascular control system

33

compensates by reducing sympathetic vasoconstriction (reduced renin-angiotensin
system activity and other mechanisms that help to maintain homeostasis). During
HDT in rats, I anticipated an initial decrease in impedance in the chest (increase in
fluid volume) as blood gathers in the central compartment Impedance in the
abdomen should increase (decrease in fluid volume) as blood shifts towards the
central venous compartment due to gravity. Although these are expected findings, a
number of studies done in humans reported that stroke volume, cardiac output and
thoracic admittance show minimal or no change in response to HDT.79'92'93
Presumably this is because the central venous compartment 1) has relatively low
compliance, 2) is maintained near maximal volume even in upright humans, and 3)

is rapidly emptied of excess volume by cardiac pumping.

W:

The bioimpedance method will detect appropriate directional changes in central

and peripheral venous compartment blood volume during HUT and HDT in rats.

12mm:

Electrodes for segmental bioimpedance measurements were implanted in a rat as
described under General Methods. While under general anesthesia, the
instrumented animal was strapped supine to a tilt table board. It was maintained in
a supine position during a 10-minute control period, to establish steady state
bioimpedance values. This was followed by ~90° HUT for 5 minutes. The animal

was returned to the supine position to re-establish initial steady values, which

34

followed a ~90° HDT for 5 minutes. Only bioimpedance was recorded during this

protocol.

Results:
The results for HUT are presented in figure 3. During HUT, estimated fluid volume

decreased in the thoracic compartment while it simultaneously increased in the
abdominal compartment. When the rat was returned to the supine position,
estimate fluid volumes returned to their control values within about 2 minutes. The
outcome for HDT is shown in figure 4. During HDT, estimated fluid volume
increased in the thoracic compartment while it simultaneously decreased in the
abdominal compartment. When the rat was returned to the supine position,
estimate fluid volumes had not yet returned to their control values within 2-3
minutes. It is not possible to determine from these measurements the absolute
volumes of fluid moving in and out of the compartments during tilting. However, to
provide additional information on the sensitivity of the bioimpedance technique,
Figure 5 shows higher gain recordings of abdominal and thoracic impedance in the
animal in the supine position. Note that a steady, rhythmic impedance recording
was obtained that which is consistent with the phases of the animal’s inspiration

and expiration.

The bioimpedance measurements for HUT and HOT in this study was consistent
with the volume shifts expected during postural tilt, as previously described. From

the literature, stroke volume is shown to differ by ~8% during inspiration and

35

expiration94 and the normal central blood volume in a 330g rat is ~4.0 ml.95 Using
this difference as an index of alterations in central blood volume during
respiration—and the data in Figure 5—allows me to conclude that the
bioimpedance method is able to detect within-animal changes in compartmental
volume of less than 0.3 ml. Since the volume of blood that can be transferred in and
out of the splanchnic and abdominal compartments is on the order of 2-3 ml in the
rat (i.e. 10-15% of total blood volume), the bioimpedance method has adequate
sensitivity for the proposed studies. From these findings, I conclude that I have
established a bioimpedance technique that can be used in a conscious animal for
repeated (or continuous) measurements of physiologically relevant changes in

compartmental fluid volume.

36

II. Specific Aim 2: To use bioimpedance to estimate blood volume changes
in the abdomen (peripheral venous compartment) and the chest
(central venous compartment) during the onset of DOCA-salt

hypertension in conscious rats.

W:

The natural progression of essential hypertension has been shown to be associated
with an increase in central blood volume.”'”'17 This excess of blood in the central
compartment could increase cardiac filling pressure; and by the Frank-Starling
mechanism cause more blood to be ejected into the low compliance arterial system.
This would result is modest arterial distension and an increase in arterial pressure.
Two mechanisms, as described earlier, can increase blood in the central venous
compartment resulting in an elevation in blood pressure during hypertension
development”:20 One mechanism is by increasing total blood volume contained in
the circulatory system. This could be achieved by an alteration in the pressure-
natriuresis mechanism, thereby causing the kidneys to conserve salt and water,
resulting in an increase in total and central blood volume.”25 This is the
prototypical explanation for DOCA-salt hypertension, i.e. an increase in total body

fluid producing increased arterial pressure!“5

The other possible mechanism is by a decrease in peripheral venous capacitance,
thereby mobilizing stored blood towards the central compartment (heart and lungs)
and into the arteries. This transfer of additional blood from peripheral to central
venous compartment would result in an elevation in arterial pressure without a

change in total blood volume.14:16'”' Vasoconstriction of splanchnic arteries and

37

veins can greatly contribute to this redistribution of blood volume.”21 Recent
studies using the DOCA-salt model, as previously described, suggest that the onset of
hypertension may also occur via this mechanism.” However, to date there is no
direct evidence for blood volume redistribution during the development of DOCA-

salt hypertension.

Therefore, bioimpedance was used in the DOCA-salt rat model to further explore
changes in blood volume within the peripheral and central venous compartment
during hypertension development. Measurements were obtained daily in conscious
rats. The chance to determine estimated compartmental blood volume during the
transition from normal arterial pressure to hypertension and back to normal
pressure is important to test my central hypothesis, i.e. a decrease in peripheral
venous capacitance and a redistribution of blood volume, which is particularly
important in the development of DOCA-salt hypertension.

W5:

During the onset of DOCA-salt hypertension, peripheral venous capacitance is
reduced and blood volume is redistributed from the abdomen (peripheral venous
compartment) to the chest (central venous compartment) and ultimately into the

arterial circulation, thereby increasing arterial pressure.

mm:

Impedance electrodes were implanted in two groups of rats, DOCA-treated and
SHAM. All rats underwent uninephrectomy and had a radiotelemeter inserted for

blood pressure measurement, as described in the Methods section. Bioimpedance

38

was individually recorded in each rat daily for 20 minutes during the study as
described in the methods. Blood pressure was recorded continuously throughout

the study. The protocol for this study is outlined below in figure 2.

 

 

 

 

 

 

'r-Uninephrectomy 33:33?
.Telemeter ' (150 mglkg SC) DOCA
. . . group
:Electrode Implantation I DOC A group i switched to
switched to saline ». dmmed H20

 

Acclimatization :Recovery’ Control Saline: DOCA salt 0'5”“

H20 ,
-7 0 5 10 13 18 21
Days

y-—.—.——. <

s 4..

Figure 2: The protocol for the development of DOCA-salt hypertension in SHAM and
DOCA-salt treated rats.

Restart:
A total of 15 rats were studied in two groups (SHAM and DOCA). The mean arterial

pressure response for both groups is shown in figure 6A. SHAM rats exhibited a
stable blood pressure throughout the protocol. High salt intake due to saline
drinking caused a small, but non-significant increase in MAP (~7 mmHg). The
combination of DOCA and salt caused a rise in MAP that was statistically significant
by days 4-5 of DOCA treatment. MAP reverted back to control values within 3 days

after the rats were switched back to distilled water to drink.

39

Figure 68 represents the estimated fluid volume in the thorax for both groups.
Neither the DOCA nor SHAM rats showed a detectable change in thoracic fluid

volume throughout the course of this study.

The estimated fluid volume in the abdomen for SHAM and DOCA is presented in
figure 6C. SHAM rats showed no change in fluid volume in the abdomen during the
course of the study. High salt intake alone due to saline drinking caused no
detectable change in abdominal fluid volume. However, a statistically significant
decrease in abdominal fluid volume was detected as early as one day after beginning
combined treatment with DOCA-salt; this reached statistical significance on days 4-5
of combined treatment Abdominal fluid volume returned to near initial control
period values when the DOCA-salt treated rats were switched to distilled water to

dfink

Conditions:

Blood volume redistribution occurred early during the development of DOCA-salt
hypertension. This was shown by a decrease in estimated abdominal fluid volume,
suggesting a decrease in peripheral venous capacitance. When DOCA-treated rats
were switched back to distilled water, representing a recovery to a normal salt diet,
both mean arterial pressure and abdominal estimated fluid volume reverted back to
initial control values. These findings support the hypothesis that peripheral venous

capacitance decreases during the onset of hypertension.

40

Interestingly, during the onset of hypertension there was no detectable change in
estimated fluid volume in the chest (central venous compartment). This finding did
not conform to the expected pattern of a shift in peripheral venous blood to the
central venous compartment Nevertheless, the results were not a total surprise,
considering the previously described human data on head down tilt, where no
change in thoracic fluid volume was seen.92 One hypothesis for this finding is that
the bioimpedance technique is not sensitive enough to detect small changes in fluid
content within the chest region. However, in light of the data from Specific Aim 1,
this possibility seems unlikely considering the high sensitivity of the bioimpedance
technique to detect volume changes with regards to breathing as observed in figure

5.

Another alternative hypothesis is that the lack of measureable blood volume change
in the chest is due to the heart. This sensitive “demand pump” rapidly transfers
most of the blood volume redistributed from the abdominal region into the arteries
and other parts of the circulation, possibly making impedance measurements
undetectable.

In summation, the overall results are consistent with the idea of blood volume
redistribution, out of the abdominal region into the central circulation, as a
contributing cause in hypertension development, but do not prove a cause-and-

effect relationship.

41

III. Specific Aim 3: Determine if activation of the sympathetic nervous
system contributes to the redistribution of blood volume during the
development of DOCA-salt hypertension.

133mm:

Splanchnic veins and venules are innervated by the SNS.52'53 Sympathetic
venoconstriction of these vessels reduces venous blood volume and overall vascular
capacitancefi‘ilo-21 This could result in a redistribution of stored blood into the
central circulation and ultimately increase arterial pressure. In addition, in DOCA-
salt hypertension there is an increase in splanchnic venomotor tone mediated by
increased sympathetic activity.”54 All the above indicates that increased SNA to the
splanchnic circulation could be one mechanism involoved in DOCA-salt mediated
hypertension. The majority of sympathetic innervation to the splanchnic
vasculature in the rat is through the celiac plexus.52 Surgical removal of this plexus
(celiac ganglionectomy, CGX) was used to investigate the contribution of
sympathetic nerve activation to the redistribution of blood volume during the

development of DOCA-salt hypertension in the rat.

W:

Selectively removing sympathetic nerve activity to the splanchnic circulation by CGX
will attenuate blood volume redistribution and the development of DOCA-salt

hypertension.

42

2mm:
Surgical CGX or SHAM-operation (SGX) were performed during the same surgery as

the impedance electrodes, radiotelemeter and uninephrectomy. Animals were given
7 days to recover before the start of the experiment. The protocol and
measurements made in this study were otherwise the same as rats in the DOCA
group in Specific Aim 2 shown in figure 2. At the conclusion of the study, the
splanchnic organs represented by the liver, spleen, small intestines and right kidney
were harvested and measured for norepinephrine content by HPLC to confirm

effective splanchnic denervation.

Ream:

A total of 14 rats were studied in two groups. Tissue norepinephrine content of the
splanchnic organs, verifying successful celiac ganglionectomy, is shown in figure 7.
Celiac ganglionectomy nearly abolished tissue norepinephrine content in the spleen,
small intestine and liver. The effect of CGX on MAP responses to DOCA-salt is
presented in figure 8A. First, rats with CGX had reduced MAP compared to sham
rats throughout the protocol. Second, both CGX and SGX rats exhibited modest and
similar increases in MAP (~5 mmHg) when on high salt intake due to drinking
saline. However, when both groups were given DOCA and salt, MAP increase was
statiscially significant in SGX on day 2 through 5, but only minimally in CGX, rats.
Switching back to distilled water to drink reduced MAP back towards control period
values in SGX rats, and minimally reduced MAP in CGX rats. Data on estimated
thoracic fluid volume are shown in figure 88. There was no observed change in

thoracic fluid volume for either group throughout the protocol. Data on estimated

43

abdominal fluid volume are presented in figure 8C. There was no change observed
in estimated abdominal fluid volume for either group throughout the study. This is

consistent with my hypothesis for CGX rats but a surprise for the SGX rats.

Because of the very unexpected lack of change in the abdominal fluid volume data in
rats that underwent abdominal surgery (CGX and SGX), I compared their data to
those from rats that did not have abdominal surgery (Figure 9). Based on our
bioimpedance measurements, abdominal fluid volume in rats that underwent
abdominal surgery for celiac ganglionectomy or sham operation appeared to be
lower than in rats that did not have abdominal surgery. This was an unexpected
finding. Thoracic fluid volume in rats that underwent abdominal surgery was higher
than in rats that did not have abdominal surgery. This was also an unexpected

finding.

Conclusion:
From this study, I conclude that CGX selectively disrupts sympathetic innervation to

the splanchnic organs and impairs development of DOCA-salt hypertension. In
studies described under Specific Aim 2, a decrease in estimated abdominal blood
volume occurred with the development of DOCA-salt hypertension. In this study,
DOCA-salt treated rats with CGX had no detectable change in estimated abdominal
fluid volume. This was expected according to my hypothesis, because removing
sympathetic innervation by CGX should prevent constriction of the splanchnic veins
and the resulting redistribution of blood volume towards the heart and arteries.

However, the sham surgery rats that were treated with DOCA-salt also showed no

44

change in abdominal fluid volume. This was a surprise. What was expected was a
decrease in abdominal fluid volume corresponding with the observed increase in
arterial pressure, as I reported in studies under Specific Aim 2. From these findings
it appears that abdominal surgery, at least in the SXG rats, is in some way, altering
blood volume regulation. The surgery, (abdominal incision along with blunt
dissection of the celiac plexus) to disrupt splanchnic inveration is relatively minor,
however, the process of locating the celiac plexus is fairly invasive, as all abdominal
organs (stomach, intestines, spleen) need to be carefully retracted and excised. It is
possible that tissue damage can occur even with the up most caution. Thus, the
addition of pro-inflammatory and anti-inflammatory mediators and the governing
inflammation process due to abdominal surgery and manipulation of the
gastrointestinal organs could possibly explain the unanticipated results in the sham
surgical animals. In a previous study, simple laparotomy (abdominal incision) and
gentle handling of the stomach, intestines and spleen resulted in microvascular
changes within these tissues.95-98 These changes included an increase in vascular
permeability, plasma extravasation, as blood moved from the capillaries to the
surrounding injured tissues. 96.93 The abdominal organs being handled during
surgery are representative of the splanchnic organs (small intestine, spleen, and
liver) and are reported to be most sensitive to surgical stress.98 In fact, surgical
stress has shown to increase vascular resistance within the vasculature of these
splanchnic organs, but not uniformily‘”, which could possibly explain the initial
differences seen in the control period blood volume observed in the abdomen and

thorax of surgery rats compared to non surgery rats in figure 9. A decrease in

45

arterial to venous blood transfer due to an increase in vascular resistance could
result in a redistribution of splanchnic venous blood towards the heart to maintain
the cardiac volume and filling pressure, but not enough to cause hypertension.
From these findings and my results, I conclude abdominal surgery and the handling
of the gastrointestinal organs alters the regulation of splanchnic vascular

capacitance and blood volume.

To further investigate the surgical phenomena, plasma volume was measured in one
DOCA-salt treated, SHAM-CGX rat, i.e. a rat subjected to abdominal surgery. The
results are shown in figure 10. The development of hypertension in this rat (that
underwent only abdominal surgery) was associated with an increase in total blood
volume. Rats without abdominal surgery do not generally have an increase in
plasma volume during DOCA-salt hypertension development, which is supported by
the abdominal redistribution results in Specific Aim 2. This preliminary finding
contributes to the idea that abdominal surgery significantly affects blood volume
regulation. More experiments in DOCA-salt treated rats are needed to confirm and

explain this abdominal surgery phenomenon.

In addition, plasma volume was measured in both surgery and non- surgery animals
during the control period before the initiation of the DOCA-salt protocol. This data
is shown in figure 11. In these preliminary results, abdominal surgery animals had a
10% lower plasma volume during the control period than non-abdominal surgery

animals. This confirms with previous studies that plasma volume is decreased in

46

rats that undergo laparotomy and gastrointestinal organ manipulation96'97-99 The
loss of extracellular fluid is largely due to an internal redistribution i.e. plasma
extravasation98 (movement of plasma from the capillaries towards the surgical site)
rather than loss from the surgical site , which is minimal. These findings further
support my conclusion that abdominal surgery alone affects the control of blood

volume.

When considering all of these preliminary plasma volume results, the data suggest
that in animals that undergo abdominal surgery an increased total circulating blood
volume contributes to the development of DOCA-salt hypertension. In DOCA-
treated animals that do not undergo abdominal surgery, the early development of
DOCA-salt hypertension appears to be due by a redistribution of blood volume.
Overall, it is clear that decreased splanchnic blood volume per se is not necessary for

development of DOCA-salt hypertension.

0 "D' . IE I .

The onset of hypertension has often been characterized by an increase in central
blood volume. This can be achieved by either an increase in total blood volume or
by a redistribution of blood volume into the central compartment (heart and lungs).
The purpose of this study was to investigate, in the DOCA-salt model, if a
redistribution of blood volume caused by a decrease in peripheral venous

capacitance contributes to this finding. The results of this study did confirm a

47

statistically significant reduction in the abdominal blood volume during the
development of arterial pressure elevation. This supports my hypothesis that a
redistribution of blood volume is important in the development of hypertension.
However, when exploring the possible mechanisms contributing to this
redistribution of peripheral venous volume, presumably by sympathetically
mediated vasoconstriction of splanchnic veins, there were unexpected findings. For
example, animals that had undergone CGX, a procedure that requires an incision in
the abdomen (laparotomy) and handling of the abdominal organs to locate and
denervate the splanchnic vasculature, showed expected results of attenuating
increases in arterial pressure and no change in abdominal blood volume. At the
same time, the control group (SGX/DOCA), which also underwent a similar
abdominal surgery but no CGX, showed very unexpected results of no change in
abdominal blood volume despite becoming hypertensive. It appears that abdominal

surgery, at least for SGX, causes an alteration in blood volume regulation.

During surgery and manipulation of the gastrointestinal organs (stomach, intestines,
spleen and often liver), it is possible that unforeseen tissue damage could result in
local inflammation, leading to the accumulation of both cellular (neutrophils and
leukocytes) and chemical mediators (such as histamine, pro-inflammatory cytokines
and inducible nitric oxide) at the surgical site, resulting in a change in the
microcirculation of these surgically manipulated tissues, as seen in previous

abdominal surgery rats.96.98

48

One possible explanation for the alteration in the microvascular after surgery has
been linked to nitric oxide (NO), which can be formed continuously in the vascular
endothelium and neuronal tissue by the constitutive NO synthase (endothelial NO
synthase and neuronal NO synthase, respectively).100 Endogenous NO is known to
play a key part in maintaining microvascular integrity during normal physiological
circumstances.101 It is important to note that NO can also be formed in blood vessels
due to various pathological conditions, such as prolonged hemorrhage and septic
shock, by the inducible form of nitric oxide synthase!”103 The physiologically
generated NO by endothelial NO synthase beneficial microcirculatory effects are
reported to attenuate vascular permeability, plasma extravasation and
inflammation)“ Therefore, rats that underwent laparotomy and gastrointestinal
handling had a reduction in endogenous NO production and increase in vascular
permabilityfi":98 When these animals were given NO synthase inhibitor, NG-nitro-L-
arginine methyl ester (L-NAME), there was an increase in plasma extravasation ,
which was made better with NO clonor,-nitroso-glutathione.96 This confirms the
importance of endogenous NO in maintaining microvascular integrity. during
surgery. But, most importantly, verifies abdominal surgery and gastrointestinal
organ manipulation disrupts the vascular endothelium by decreasing NO. While
this is only one mechanism out of many that could occur during abdominal surgery
and gastrointestinal organ handling, further investigation into selectively disrupting
the celiac plexus without the need to open the abdominal cavity and handle the

stomach and intestines would help address this surgical phenomenon.

49

In conclusion, the early development of hypertension in DOCA-salt treated animals
can be established by either a redistribution of blood volume and by an increase in
total blood volume, and is based on if surgical manipulation is involved. Overall, a
decrease in splanchnic blood volume is not required for the development of DOCA-

salt hypertension in this study

50

Bioimpedance 53
Head Up Tilt .j

A. Chest

 

 

 

 

(D
U
C A . .
g a, 24 tilt supine
m E ' l: I I" : 9'
(1.: : if" 1 " '1 Tats“.
E 3 16 I r‘ n I ' “"5...
:5 cams.» r." : ' 1‘ “WV"
CD 8 I 1
B. Abdominal
8 4e
‘ l
g ’0" _rfinwr—fiaw‘y : ‘:"I‘“~VW~
I II
E E 42 : “(3.. ‘ W 5 +3 I: r
.' -' .t. I“ .it
E 3 38 i ' 'L‘ “ “l
" tilt su ine
is: 3. p
0 1 3 4 5 (mins)

Figure 3: Chest and abdominal bioimpedance recording during a head up postural
tilt. The animal began in a supine position and was brought to a ~90° HUT and

finished in a supine position. The duration of the experiment was 5 minutes.

51

l

Bioimpedance \Yi“;

Head Down Tilt “I g

 

 

 

 

EA A. Chest m
U m I su ine
SE 36 l .p
.E 8 24 mw I l) .H. ,‘ ~ nwp.,,-a‘ 1.5 “I."1‘."""M'“"W"'triad“ :
" N” "w" M-ry.~.~.-.,~........L-.» ~~_—~—.. _ . s... —.. .t.
m 12 ' .
8 - .
gA B. Abdomlnal _ supine
‘o "’ lllt __.....,.-~-:-._..-.. mat-Am-
g E 38 I
.C 34 mw’vHIIIJm-r “La.” I. L'"""""l“' \,_,l.f*._I-|'“’”U '
E 3 ' I ,
‘1‘ o 1 2 3 4 5 (mins)

Figure 4: Chest and abdominal bioimpedance recording during a head down
postural tilt. The animal began in a supine position and was brought to ~90° HDT
and finished in a supine position. The duration of the experiment was 5 minutes.

52

Bioimpedance '+ rt»

 

 

 

Supine __
\
.VQ
g A. Chest
t:
58 7;
8E 27 4'... ,.._~., 1.. , ‘ H .--'~.
E 3 :3 I: h ‘I')"‘g"1"l‘: #113." 4 try iglr‘!‘ "":"i1"‘-|-'~:.g.lm".'g."i‘;.‘:a-1}_-I‘."“ ‘I'fllr‘ft-‘i‘ni l?” l" "flirt
'5
CD
§ 8. Abdominal breath breath
(U A
8 2 24 ,, ..
» ”with we? .; 3.- s-Iw. 3.x, (AH;- .m
Q-C "' P“ m )4“ tag IN 1"» ‘l, 1‘-
.g 8 Z W", \‘T" “l9? ‘5' 9'34..on ‘:.V’
0
CD 0 1 2 3 4 5 (mins)

Figure 5: Bioimpedance recordings during a supine position. The duration of the
experiment was 5 minutes.

53

>

 

 

135 — Control Salinerl DOCA;Tsailiné7Ii H26
W ,TWW- ,W, A ,-W a , W

t

130‘
125-
120-
115‘
110-
105- I
100- i l
95...r.'.4.....f’.

+ SHAM (n=5)

1
I
I —o— DOCA (n=9)
I
I

 

   

 

 

Mean Arterial Pressure (mmHg)

 

W

Control Saline DOCA+saline H20
W W. W. WW W W W W .W

Thoracic __.._ SHAM (n=6)

—0— DOCA (n=9)

Estimated Fluid Volume (1IZ)

 

 

 

 

 

 

 

Control I Saline

DOCA+saline
0.10 ~77” .

Abdominal

 

 

—O— DOCA (n=9)

   

|
|
i
|
|
|
|
|
|

  

|
|
j + SHAM (n=6)
|
|
|
|
I
|

  

|
I l

| I

I I o.
I { **
I

I I

I I

 

 

Estimated Fluid Volume (1IZ) “

0 1 2 3 4 5 6 7 8 910111213141516
Days

Figure 6: Mean arterial pressure (A), estimated thoracic fluid volume (B) and
estimated abdominal fluid volume (C) response in rats treated with DOCA-salt or
distilled water. * Significant difference (p<0.05) compared to control day 2.

54

800
@700
g 600
w 500
Z 400
S 300
In
.9 200
100

' SGX (n=3) '
D CGX (n=5) ¢ 990/
0
I96%
I 50% {81%
—W - , a,

Kidney Liver Sl Spleen

Figure 7: Tissue norepinephrine (NE) content in the splanchnic organs in response
to celiac ganglionectomy (CGX) and SHAM-operated (SGX) in DOCA-salt treated rats.

55

>

150

Mean Arterial Pressure (mmHg)

90

0.09

0.05

0.03

0.07

Estimated Fluid Volume (1IZ) n Estimated Fluid Volume (1IZ)

0.03

 

Control

Saline

DOCA+saline

H20

 

140 -

130 3

120 -

110 ‘

100 .

 

 

 

****

WEE/em”

 

 

 

0.08 -

0.07 -

0.06 -

0.04 *

0.06 -

0.05 .

0.04 -

 

Control

Saline ‘1 DOCA+saline

 

Thoracic

1

|
I
|
|
I
|
|
|
|
I
I
|

 

 

 

 

 

Control

: Saline IDOCA+saline

H20

 

Abdominal

 

 

 

 

012 3 4 5 6 7 8 910111213141516
DIYS

+ DOCA / sex (n=7)
—Cl— DOCA l cox (n=9)

+ DOCA/SGX (n=4)
—I:I— DOCA/CGX (n=8)

+ DOCAI SGX
—Cl— DOCA I CGX

rn=4l
In=8l

Figure 8: Mean arterial pressure (A), estimated thoracic fluid volume (B) and

estimated abdominal

fluid volume (C) response to DOCA-salt

ganglionectomized (CGX) or SHAM operated rats

56

in celiac

Estimated Fluid Volume (1IZ) n Estimated Fluid Volume (1IZ) a: Mean Arterial Pressure (mmHg) >

150

140-

130 -

120 -

110

100 <

90

Q
—l
O

.0
o
co

.0
o
a)

.0
o
A

.0
o
N

0.12 W

.0
_l
o

.0
0
on

.0
o
a)

.0
O
A

 

Control Saline DOCA+saline H20 '

 

 

 

 

 

 

 

 

Control

Thoracic

 

 

 

 

 

Control Saline IDOCA-o-saline H20 I

 

 

 

Abdominal

I I
l l W

0 1 2 3 4 5 6 7 8 9 10111213141516
Days

 

 

 

—-— DOCA/ sex (n=7)
—D— DOCA/CGX (n=9)
+ SHAM (n=5)
--0— DOCA(n=9)

+ SHAM (n=6)
—0— DOCA (n=9)

+ DOCA] sex (n=4)
—I:I— DOCA] cex (n=8)

+ SHAM (n=6)
~0— DOCA(n=9)

+ DOCA/SGX (n=4)
—Cl-- DOCA/CGX (n=8)

Figure 9: Mean arterial pressure (A) and the response of estimated thoracic (B) and
abdominal (C) fluid volume in abdominal surgery rats (CGX and SGX) and non-
abdominal surgery rats (DOCA and SHAM).

57

 

 

 

 

 

 

 

 

‘3

IE: Control Saline DOCA+salineI H20
E 180

d)

S 150. 25.3 20.1
(D

(D

2

E 140-

g 19.8

‘-

E 120- 21-8

<

5

w100.....T. 4...4....
E 012 3 4 5 6 7 8 910111213141516

Days

Figure 10: Mean arterial pressure response to DOCA-salt in SHAM operated (SGX)
rat (n=1). The numbers in the box represent plasma volume (ml).

58

l)
l\) [\J (.0 O)
O 01 0 UI

_x
O

Plasma volume (m
tn 6%

 

 

O

Non- Surgery
surgery (0:3)
(n=2)

Figure 11: The plasma volume results for non-abdominal and abdominal surgery
rats.

59

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